Osteoblasts are mesenchymally derived cells responsible for bone formation. Defects in osteoblast development are linked to genetic and degenerative skeletal diseases and bone tumor formation. The transcription factor Runx2 (CBFA1, AML-3) is absolutely required for osteoblast development and Runx2 mutations cause cleidocranial dysplasia (CCD). Thus, regulation of Runx2 activity is crucial for bone formation. Runx2 regulates tissue specific gene expression by binding to the DNA sequence, TGPuGGTPu, to either activate or repress transcription. Our long-term objective is to identify molecular mechanisms that effect Runx2 activity during osteoblast differentiation. In many non-osteoblast cells, associations between lineage-required transcription factors and proteins possessing either acetyltransferase or deacetylase activity regulate gene expression and cell differentiation. Thus, histone acetyltransferase (HAT) activity is associated with enhanced gene transcription, while histone deacetylase (HDAC) activity is linked to transcriptional repression. The specific aims of this project focus on identifying the mechanisms of Runx2 transcriptional repression. Our preliminary data indicate that Runx2 contains multiple repression domains, some of which interact with proteins associated with HDAC activity. The central hypothesis to be tested in these studies is that associations between Runx2 and proteins that modify chromatin structure by removing acetyl groups from histones is crucial for transcriptional repression of Runx2-targeted osteoblast-specific genes. The proposed studies will define all repression domains in Runx2 and examine interactions between Runx2 and specific HDACs and HDAC-associated co-repressors. We will also determine the roles of these factors in osteoblast-specific gene expression and differentiation. It is believed that a better understanding of the molecular mechanisms regulating Runx2 activity during osteoblast differentiation will lead to novel therapies for individuals with genetic and degenerative bone diseases or bone tumors caused by osteoblast differentiation defects.